US10172910B2 - Method of preventing or reducing the incidence of acute urinary retention - Google Patents

Method of preventing or reducing the incidence of acute urinary retention Download PDF

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US10172910B2
US10172910B2 US15/222,365 US201615222365A US10172910B2 US 10172910 B2 US10172910 B2 US 10172910B2 US 201615222365 A US201615222365 A US 201615222365A US 10172910 B2 US10172910 B2 US 10172910B2
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leu
peptide
ser
arg
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US20180028597A1 (en
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Paul Averback
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Nymox Corp
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Nymox Corp Bahamas
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Priority to PCT/IB2017/054277 priority patent/WO2018020355A1/en
Priority to CA3032254A priority patent/CA3032254C/en
Priority to ES17746197T priority patent/ES2891544T3/es
Priority to KR1020197006132A priority patent/KR102489731B1/ko
Priority to MX2019001209A priority patent/MX2019001209A/es
Priority to EP17746197.7A priority patent/EP3490580B1/en
Priority to RU2019105553A priority patent/RU2721538C1/ru
Priority to JP2019504756A priority patent/JP6768140B2/ja
Priority to CN201780046897.3A priority patent/CN109562141A/zh
Priority to AU2017304100A priority patent/AU2017304100B2/en
Priority to BR112019001750-0A priority patent/BR112019001750A2/pt
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/18—Sulfonamides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers

Definitions

  • the embodiments include methods of preventing acute urinary retention using compositions containing compounds based on small peptides and a pharmaceutically acceptable carrier.
  • the methods include, but are not limited to, administering the compositions intramuscularly, orally, intravenously, intraprostatically, intraperitoneally, intracerebrally (intraparenchymally), intracerebroventricularly, intralesionally, intraocularly, intraarterially, intrathecally, intratumorally, intranasally, topically, transdermally, subcutaneously, or intradermally to patients in need thereof, wherein those patients to whom the compositions have been administered have a dramatic decrease in incidence of acute urinary retention (AUR).
  • AUR acute urinary retention
  • Benign prostatic hyperplasia is common in older men, with symptoms that impact quality of life, including interference with activities and perception of well being. BPH can be progressive, with risk of urinary retention, infections, bladder calculi and renal failure. Although many men with mild to moderate symptoms do well without intervention, bothersome symptoms and complications can progress in others, leading to medical therapy or surgery.
  • Acute urinary retention may be classified as either spontaneous or precipitated.
  • Spontaneous acute urinary retention is often considered by patients to be the most serious outcome of BPH.
  • Spontaneous acute urinary retention is an episode of acute urinary retention that is due to BPH and is not tied to a precipitating event.
  • the 5 ⁇ -reductase inhibitor finasteride has been shown to be effective in treating BPH and in significantly reducing spontaneous acute urinary retention in patients with BPH. Andersen et al., Urology, 49(6), 839-845 (1997).
  • Precipitated acute urinary retention is an episode of acute urinary retention that is precipitated by at least one of the following factors: anesthesia or surgery within 72 hours; a precipitating medical event such as stroke or congestive heart failure; a medical condition such as prostatitis or urinary tract infection; or ingestion of medication or drugs known to precipitate retention, e.g., pseudoephedrine hydrochloride, cold medicine, pain medication such as narcotics or sedatives, or benadryl.
  • a precipitating medical event such as stroke or congestive heart failure
  • a medical condition such as prostatitis or urinary tract infection
  • ingestion of medication or drugs known to precipitate retention e.g., pseudoephedrine hydrochloride, cold medicine, pain medication such as narcotics or sedatives, or benadryl.
  • Benign overgrowths of tissue are abnormalities in which it is desirable to remove cells from an organism.
  • Benign tumors are cellular proliferations that do not metastasize throughout the body but do, however, cause disease symptoms. Such tumors can be lethal if they are located in inaccessible areas in organs such as the brain.
  • Surgery often is the first step in the treatment of cancer.
  • the objective of surgery varies. Sometimes it is used to remove as much of the evident tumor as possible, or at least to “debulk” it (remove the major bulk(s) of tumor so that there is less that needs to be treated by other means).
  • surgery may also provide some symptomatic relief to the patient. For instance, if a surgeon can remove a large portion of an expanding brain tumor, the pressure inside the skull will decrease, leading to improvement in the patient's symptoms.
  • tumors are amenable to surgery. Some may be located in parts of the body that make them impossible to completely remove. Examples of these would be tumors in the brainstem (a part of the brain that controls breathing) or a tumor which has grown in and around a major blood vessel. In these cases, the role of surgery is limited due to the high risk associated with tumor removal.
  • Hodgkin's lymphoma a cancer of the lymph nodes that responds very well to combinations of chemotherapy and radiation therapy. In Hodgkin's lymphoma, surgery is rarely needed to achieve cure, but almost always used to establish a diagnosis.
  • Chemotherapy is another common form of cancer treatment. Essentially, it involves the use of medications (usually given by mouth or injection) which specifically attack rapidly dividing cells (such as those found in a tumor) throughout the body. This makes chemotherapy useful in treating cancers that have already metastasized, as well as tumors that have a high chance of spreading through the blood and lymphatic systems but are not evident beyond the primary tumor. Chemotherapy may also be used to enhance the response of localized tumors to surgery and radiation therapy. This is the case, for example, for some cancers of the head and neck.
  • Chemotherapy is administered to patients in a variety of ways. Some include pills and others are administered by an intravenous or other injection. For injectable chemotherapy, a patient goes to the doctor's office or hospital for treatment. Other chemotherapeutic agents require continuous infusion into the bloodstream, 24 hours a day. For these types of chemotherapy, a minor surgical procedure is performed to implant a small pump worn by the patient. The pump then slowly administers the medication. In many cases, a permanent port is placed in a patient's vein to eliminate the requirement of repeated needle sticks.
  • Benign tumors and malformations also can be treated by a variety of methods including surgery, radiotherapy, drug therapy, thermal or electric ablation, cryotherapy, and others.
  • benign tumors do not metastasize, they can grow large and they can recur.
  • Surgical extirpation of benign tumors has all the difficulties and side effects of surgery in general and oftentimes must be repeatedly performed for some benign tumors, such as for pituitary adenomas, meningeomas of the brain, prostatic hyperplasia, and others.
  • some patients who receive non-surgical treatment to ameliorate the symptoms caused by benign tumors still require subsequent invasive surgical intervention.
  • Lepor “Medical Treatment of Benign Prostatic Hyperplasia,” Reviews in Urology, Vol. 13, No. 1, pp. 20-33 (2011), discloses a variety of studies of the efficacy of drug therapies in treating BPH, and the need for subsequent invasive surgical treatment.
  • Blockade of testicular androgen secretion by surgical or medical (LHRH agonist) castration is known to decrease prostatic size (Auclair et al., Biochem. Biophys. Res. Commun. 76: 855-862, 1977; Auclair et al., Endocrinology 101: 1890-1893, 1977; Labrie et al., Int. J. Andrology, suppl. 2 (V. Hansson, ed.), Scriptor Publisher APR, pp. 303-318, 1978; Labrie et al., J.
  • U.S. Pat. No. 3,423,507 discloses the use of the antiandrogen cyproterone acetate (1 ⁇ , 2 ⁇ -methylene-6-chloro-17 ⁇ -acetoxy-6-dehydroprogesterone) for the treatment of benign prostatic hyperplasia.
  • Pure antiandrogens U.S. Pat. No. 4,329,364 cause an increase in testosterone secretion, which can result in a higher degree of aromatization into estrogens, a situation expected from current knowledge to have negative effects on prostatic hyperplasia (Jacobi et al., Endocrinology 102: 1748-1755, 1978).
  • the antiestrogen Tamoxifen has been shown to improve steroid-induced benign prostatic hyperplasia in the dog (Funke et al., Acta Endocrinol. 100: 462-472, 1982).
  • Administration of the antiestrogen Tamoxifen in association with the steroidal antiandrogen cyproterone acetate in patients suffering from benign prostatic hyperplasia showed beneficial effects on the symptoms of the disease (Di Silverio et al., in Ipertrofia Prostatica Benigna (F. Di Silverio, F. Neumann and M. Tannenbaum, eds), Excerpta Medica, pp. 117-125, 1986).
  • U.S. Pat. No. 4,310,523 it is proposed that a combination of an antiandrogen and an antiestrogen is effective for the prophylaxis and/or therapy of benign prostatic hyperplasia. Tamoxifen, however, has intrinsic estrogenic activity which limits its effectiveness.
  • Estrogen formation resulting from aromatization of androgens occurs at several sites.
  • aromatization of androgens has been demonstrated in the testis, adipose and muscle tissue, skin, liver, brain and prostate (Schweikert et al., J. Clin. Endocrinol. Metab. 40: 413-417, 1975; Folker and James, J. Steroid Biochem. 49: 687-690, 1983; Longcope et al., J. Clin. Endocrinol. Metab. 46: 146-152, 1978; Lacoste and Labrie, unpublished data; Stone et al., The Prostate 9: 311-318, 1986; Stone et al., Urol. Res.
  • U.S. Pat. No. 4,472,382 discloses treatment of BPH with an antiandrogen and certain peptides which act as LH-RH agonists.
  • U.S. Pat. No. 4,596,797 discloses aromatase inhibitors as a method of prophylaxis and/or treatment of prostatic hyperplasia.
  • U.S. Pat. No. 4,760,053 describes a treatment of certain cancers which combines an LHRH agonist with an antiandrogen and/or an antiestrogen and/or at least one inhibitor of sex steroid biosynthesis.
  • U.S. Pat. No. 4,775,660 discloses a method of treating breast cancer with a combination therapy which may include surgical or chemical prevention of ovarian secretions and administering an antiandrogen and an antiestrogen.
  • U.S. Pat. No. 4,659,695 discloses a method of treatment of prostate cancer in susceptible male animals including humans whose testicular hormonal secretions are blocked by surgical or chemical means, e.g. by use of an LHRH agonist, which comprises administering an antiandrogen, e.g. flutamide, in association with at least one inhibitor of sex steroid biosynthesis, e.g. aminoglutethimide and/or ketoconazole.
  • an antiandrogen e.g. flutamide
  • at least one inhibitor of sex steroid biosynthesis e.g. aminoglutethimide and/or ketoconazole.
  • BPH is caused by increased activity of both androgens and estrogens. Because of such a dual etiology of BPH, proposed hormonal therapies have been less than satisfactory and have all been unpredictable while, frequently, causing unacceptable side-effects. Moreover, the prior art treatment seldomly resulted in a decrease in prostatic volume above about 20 to 30% with inconsistent effects on the symptomatology (Scott and Wade, J. Urol. 101: 81-85, 1969; Caine et al., J. Urol. 114: 564-568, 1975; Peters and Walsh, New Engl. J. Med. 317: 599-604, 1987; Gabrilove et al., J. Clin. Endocrinol. Metab.
  • NTP peptides including a specific peptide described by the amino acid sequence Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Gludle-Lys-Arg-Cys-Leu (SEQ ID NO: 66), are capable of preventing or reducing the incidence of acute urinary retention.
  • NTP peptides including a specific peptide described by the amino acid sequence Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Gludle-Lys-Arg-Cys-Leu (SEQ ID NO: 66), either alone or in combination with an additional active agent capable of treating and/or killing unwanted cellular proliferations in mammals, provide an unexpected improvement in patients suffering from or susceptible to developing acute urinary retention.
  • Some embodiments are directed to methods of preventing or reducing the incidence of acute urinary retention in mammals comprising administering to a mammal in need thereof a therapeutically effective amount of a composition comprising an NTP peptide, either alone or in combination with at least one additional active agent capable of treating and/or killing unwanted cellular proliferations in mammals.
  • the compositions can be administered intramuscularly, orally, intravenously, intraperitoneally, intracerebrally (intraparenchymally), intracerebroventricularly, intratumorally, intralesionally, intradermally, intrathecally, intranasally, intraocularly, intraarterially, topically, transdermally, via an aerosol, infusion, bolus injection, implantation device, sustained release system etc.
  • the NTP peptides can be expressed in vivo by administering a gene that expresses the NTP peptides, by administering a vaccine that induces such production or by introducing cells, bacteria or viruses that express the peptide in vivo, because of genetic modification or otherwise.
  • administering a composition comprising an NTP peptide either alone or in combination with at least one additional active agent capable of treating and/or killing unwanted cellular proliferations in mammals reduces the incidence of acute urinary retention by more than 10%, when compared to administering a control composition that does not contain an NTP peptide.
  • a host cell includes a plurality of such host cells
  • an antibody is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
  • Amino acids and amino acid residues described herein may be referred to according to the accepted one or three-letter code provided in the table below.
  • NTP peptide refers to peptides comprising amino acid sequences corresponding to at least a part of the amino acid sequence of Neural Thread Proteins or to fragments of Neural Thread Proteins and includes homologues, derivatives, variants, fusion proteins, and peptide mimetics of such peptides unless the context indicates otherwise.
  • the expression “NTP peptide” also refers to a peptide or other composition of matter claimed in one or more of the following U.S. patent application Ser. No. 14/808,713, filed Jul. 24, 2015, entitled: METHODS OF REDUCING THE NEED FOR SURGERY IN PATIENTS SUFFERING FROM BENIGN PROSTATIC HYPERPLASIA; U.S. patent application Ser. No.
  • SEQ ID NO. 1 MEFSLLLPRLECNGA or Met-Glu-Phe-Ser-Leu-Leu-Leu-Pro-Arg-Leu-Glu- Cys-Asn-Gly-Ala 2)
  • SEQ ID NO. 2 GAISAHRNLRLPGSS or Gly-Ala-Ile-Ser-Ala-His-Arg-Asn-Leu-Arg-Leu- Pro-Gly-Ser-Ser 3)
  • SEQ ID NO. 3 DSPASASPVAGITGMCT or Asp-Ser-Pro-Ala-Ser-Ala-Ser-Pro-Val-Ala-Gly- Ile-Thr-Gly-Met-Cys-Thr 4) SEQ ID NO.
  • SEQ ID NO. 29 SLLSSWDYRR or Ser-Leu-Leu-Ser-Ser-Trp-Asp-Tyr-Arg-Arg 30) SEQ ID NO. 30: SSWDY or Ser-Ser-Trp-Asp-Tyr 31) SEQ ID NO. 31: SSWDYRR or Ser-Ser-Trp-Asp-Tyr-Arg-Arg 32) SEQ ID NO.
  • SEQ ID NO. 60 PASASPVAGITGM or Pro-Ala-Ser-Ala-Ser-Pro-Val-Ala-Gly-Ile-Thr- Gly-Met 61)
  • SEQ ID NO. 61 PASASQVAGTKDM or Pro-Ala-Ser-Ala-Ser-Gln-Val-Ala-Gly-Thr-Lys- Asp-Met 62) SEQ ID NO.
  • SSRFRFWGALVCSMD Ser-Ser-Arg-Phe-Arg-Phe-Trp-Gly-Ala-Leu-Val- Cys-Ser-Met-Asp 76)
  • SEQ ID NO. 76 SCRFSRVAVTYRFIT or Ser-Cys-Arg-Phe-Ser-Arg-Val-Ala-Val-Thr-Tyr- Arg-Phe-Ile-Thr 77)
  • SEQ ID NO. 77 LLNIPSPAVWMARNT or Leu-Leu-Asn-Ile-Pro-Ser-Pro-Ala-Val-Trp-Met- Ala-Arg-Asn-Thr 78) SEQ ID NO.
  • MVVTLKSSLVLLLCLT or Met-Trp-Thr-Leu-Lys-Ser-Ser-Leu-Val-Leu-Leu- Leu-Cys-Leu-Thr 86)
  • SEQ ID NO. 86 CSYAFMFSSLRQKTS or Cys-Ser-Tyr-Ala-Phe-Met-Phe-Ser-Ser-Leu-Arg- Gln-Lys-Thr-Ser 87)
  • SEQ ID NO. 87 EPQGKVPCGEHFRIR or Glu-Pro-Gln-Gly-Lys-Val-Pro-Cys-Gly-Glu-His- Phe-Arg-Ile-Arg 88) SEQ ID NO.
  • LHHIDSISGVSGKRMF Leu-His-His-Ile-Asp-Ser-Ile-Ser-Gly-Val-Ser- Gly-Lys-Arg-Met-Phe 92)
  • SEQ ID NO. 92 EAYYTMLHLPTTNRP or Glu-Ala-Tyr-Tyr-Thr-Met-Leu-His-Leu-Pro-Thr- Thr-Asn-Arg-Pro 93) SEQ ID NO.
  • SEQ ID NO. 95 SNSHSHPNPLKLHRR or Ser-Asn-Ser-His-Ser-His-Pro-Asn-Pro-Leu-Lys- Leu-His-Arg-Arg 95) SEQ ID NO. 95: SHSHNRPRAYILITI or Ser-His-Ser-His-Asn-Arg-Pro-Arg-Ala-Tyr-Ile- Leu-Ile-Thr-Ile 96) SEQ ID NO.
  • 96 LPSKLKLRTHSQSHH or Leu-Pro-Ser-Lys-Leu-Lys-Leu-Arg-Thr-His-Ser- Gln-Ser-His-His 97)
  • SEQ ID NO. 97 NPLSRTSNSTPTNSFLMTSSKPR or Asn-Pro-Leu-Ser-Arg-Thr-Ser-Asn-Ser-Thr-Pro- Thr-Asn-Ser-Phe-Leu-Met-Thr-Ser-Ser-Lys-Pro- Arg 98) SEQ ID NO.
  • SEQ ID NO. 99 LLSLALMINFRVMAC or Leu-Leu-Ser-Leu-Ala-Leu-Met-Ile-Asn-Phe-Arg- Val-Met-Ala-Cys 100)
  • SEQ ID NO. 100 TFKQHIELRQKISIV or Thr-Phe-Lys-Gln-His-Ile-Glu-Leu-Arg-Gln-Lys- Ile-Ser-Ile-Val 101) SEQ ID NO.
  • 104 GNSSFFLLSFFFSFQ or Gly-Asn-Ser-Ser-Phe-Phe-Leu-Leu-Ser-Phe-Phe- Phe-Ser-Phe-Gln 105) SEQ ID NO. 105: NCCQCFQCRTTEGYA or Asn-Cys-Cys-Gln-Cys-Phe-Gln-Cys-Arg-Thr-Thr- Glu-Gly-Tyr-Ala 106) SEQ ID NO.
  • VECFYCLVDKAAFECVWVFYSF DT Val-Glu-Cys-Phe-Tyr-Cys-Leu-Val-Asp-Lys-Ala- Ala-Phe-Glu-Cys-Trp-Trp-Phe-Tyr-Ser-Phe-Asp- Thr 107)
  • SEQ ID NO. 107 MEPHTVAQAGVPQHD or Met-Glu-Pro-His-Thr-Val-Ala-Gln-Ala-Gly-Val- Pro-Gln-His-Asp 108) SEQ ID NO.
  • LGSLQSLLPRFKRFS Leu-Gly-Ser-Leu-Gln-Ser-Leu-Leu-Pro-Arg-Phe- Lys-Arg-Phe-Ser 109)
  • SEQ ID NO. 110 ALIKRNRYTPETGRKS or Ala-Leu-Ile-Lys-Arg-Asn-Arg-Tyr-Thr-Pro-Glu- Thr-Gly-Arg-Lys-Ser 111) SEQ ID NO.
  • SEQ ID NO. 112 KLEIKRCL or Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu 113)
  • SEQ ID NO. 113 VLSRIK or Val-Leu-Ser-Arg-Ile-Lys 114)
  • NTP peptide also preferably includes (but is not limited to) the amino acid sequences of SEQ ID NO: 1 to 116.
  • fragment refers to a protein or polypeptide that consists of a continuous subsequence of the amino acid sequence of a protein or peptide and includes naturally occurring fragments such as splice variants and fragments resulting from naturally occurring in vivo protease activity. Such a fragment may be truncated at the amino terminus, the carboxy terminus, and/or internally (such as by natural splicing). Such fragments may be prepared with or without an amino terminal methionine.
  • fragment includes fragments, whether identical or different, from the same protein or peptide, with a contiguous amino acid sequence in common or not, joined together, either directly or through a linker. A person having ordinary skill in the art will be capable of selecting a suitable fragment for use in the embodiments without undue experimentation using the guidelines and procedures outlined herein.
  • variant refers to a protein or polypeptide in which one or more amino acid substitutions, deletions, and/or insertions are present as compared to the amino acid sequence of an protein or peptide and includes naturally occurring allelic variants or alternative splice variants of an protein or peptide.
  • variant includes the replacement of one or more amino acids in a peptide sequence with a similar or homologous amino acid(s) or a dissimilar amino acid(s). There are many scales on which amino acids can be ranked as similar or homologous. (Gunnar von Heijne, Sequence Analysis in Molecular Biology, p. 123-39 (Academic Press, New York, N.Y.
  • Preferred variants include alanine substitutions at one or more of amino acid positions.
  • Other preferred substitutions include conservative substitutions that have little or no effect on the overall net charge, polarity, or hydrophobicity of the protein. Conservative substitutions are set forth in Table 2 below.
  • variants can consist of less conservative amino acid substitutions, such as selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • substitutions that in general are expected to have a more significant effect on function are those in which (a) glycine and/or proline is substituted by another amino acid or is deleted or inserted; (b) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (c) a cysteine residue is substituted for (or by) any other residue; (d) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) a residue having an electronegative charge, e.g., glutamyl or aspartyl; or (e) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not
  • variants include those designed to either generate a novel glycosylation and/or phosphorylation site(s), or those designed to delete an existing glycosylation and/or phosphorylation site(s).
  • Variants include at least one amino acid substitution at a glycosylation site, a proteolytic cleavage site and/or a cysteine residue.
  • Variants also include proteins and peptides with additional amino acid residues before or after the protein or peptide amino acid sequence on linker peptides. For example, a cysteine residue may be added at both the amino and carboxy terminals of an NTP peptide in order to allow the cyclisation of the peptide by the formation of a di-sulphide bond.
  • variant also encompasses polypeptides that have the amino acid sequence of an NTP peptide with at least one and up to 25 or more additional amino acids flanking either the 3′ or 5′ end of the peptide.
  • derivative refers to a chemically modified protein or polypeptide that has been chemically modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques, as for example, by addition of one or more polyethylene glycol molecules, sugars, phosphates, and/or other such molecules, where the molecule or molecules are not naturally attached to wild-type proteins or NTP peptides.
  • Derivatives include salts.
  • Such chemical modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given protein or polypeptide.
  • a given protein or polypeptide may contain many types of modifications. Modifications can occur anywhere in a protein or polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphoryl
  • derivatives include chemical modifications resulting in the protein or polypeptide becoming branched or cyclic, with or without branching. Cyclic, branched and branched circular proteins or polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.
  • homologue refers to a protein that is at least 60 percent identical in its amino acid sequence of an NTP peptide as determined by standard methods that are commonly used to compare the similarity in position of the amino acids of two polypeptides.
  • the degree of similarity or identity between two proteins can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H.
  • Preferred computer program methods useful in determining the identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA, Atschul, S. F. et al., J. Molec. Biol., 215: 403-410 (1990).
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol., 215: 403-410 (1990).
  • the two proteins or polypeptides for which the percent sequence identity is to be determined are aligned for optimal matching of their respective amino acids (the “matched span”, as determined by the algorithm).
  • a gap opening penalty (which is calculated as 3 times the average diagonal; the “average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1/10times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm.
  • a standard comparison matrix (see Dayhoff et al. in: Atlas of Protein Sequence and Structure, vol. 5, supp.3 for the PAM250 comparison matrix; see Henikoff et al., Proc. Natl. Acad. Sci USA, 89:10915-10919 for the BLOSUM 62 comparison matrix) also may be used by the algorithm. The percent identity then is calculated by the algorithm. Homologues will typically have one or more amino acid substitutions, deletions, and/or insertions as compared with the comparison protein or peptide, as the case may be.
  • fusion protein refers to a protein where one or more peptides are recombinantly fused or chemically conjugated (including covalently and non-covalently) to a protein such as (but not limited to) an antibody or antibody fragment like an F.sub.ab fragment or short chain Fv.
  • fusion protein also refers to multimers (i.e. dimers, trimers, tetramers and higher multimers) of peptides. Such multimers comprise homomeric multimers comprising one peptide, heteromeric multimers comprising more than one peptide, and heteromeric multimers comprising at least one peptide and at least one other protein.
  • Such multimers may be the result of hydrophobic, hyrdrophilic, ionic and/or covalent associations, bonds or links, may be formed by cross-links using linker molecules or may be linked indirectly by, for example, liposome formation
  • peptide mimetic refers to biologically active compounds that mimic the biological activity of a peptide or a protein but are no longer peptidic in chemical nature, that is, they no longer contain any peptide bonds (that is, amide bonds between amino acids).
  • peptide mimetic is used in a broader sense to include molecules that are no longer completely peptidic in nature, such as pseudo-peptides, semi-peptides and peptoids. Examples of peptide mimetics in this broader sense (where part of a peptide is replaced by a structure lacking peptide bonds) are described below.
  • peptide mimetics provide a spatial arrangement of reactive chemical moieties that closely resemble the three-dimensional arrangement of active groups in the peptide on which the peptide mimetic is based. As a result of this similar active-site geometry, the peptide mimetic has effects on biological systems that are similar to the biological activity of the peptide.
  • the peptide mimetics of the embodiments are preferably substantially similar in both three-dimensional shape and biological activity to the peptides described herein.
  • Examples of methods of structurally modifying a peptide known in the art to create a peptide mimetic include the inversion of backbone chiral centers leading to D-amino acid residue structures that may, particularly at the N-terminus, lead to enhanced stability for proteolytical degradation without adversely affecting activity.
  • An example is given in the paper “Tritriated D-ala.sup.1-Peptide T Binding”, Smith C. S. et al., Drug Development Res., 15, pp. 371-379 (1988).
  • a second method is altering cyclic structure for stability, such as N to C interchain imides and lactames (Ede et al. in Smith and Rivier (Eds.) “Peptides: Chemistry and Biology”, Escom, Leiden (1991), pp. 268-270).
  • An example of this is given in conformationally restricted thymopentin-like compounds, such as those disclosed in U.S. Pat. No. 4,457,489 (1985), Goldstein, G. et al., the disclosure of which is incorporated by reference herein in its entirety.
  • a third method is to substitute peptide bonds in the peptide by pseudopeptide bonds that. confer resistance to proteolysis.
  • the amino acid sequences of the peptides may be identical to the sequences of an peptide described above, except that one or more of the peptide bonds are replaced by a retro-inverso pseudopeptide bond.
  • the most N-terminal peptide bond is substituted, since such a substitution will confer resistance to proteolysis by exopeptidases acting on the N-terminus.
  • Further modifications also can be made by replacing chemical groups of the amino acids with other chemical groups of similar structure.
  • Another suitable pseudopeptide bond that is known to enhance stability to enzymatic cleavage with no or little loss of biological activity is the reduced isostere pseudopeptide bond (Couder, et al. (1993), Int. J. Peptide Protein Res., 41:181-184, incorporated herein by reference in its entirety).
  • amino acid sequences of these peptides may be identical to the sequences of an peptide, except that one or more of the peptide bonds are replaced by an isostere pseudopeptide bond.
  • amino acid sequences of these peptides may be identical to the sequences of an peptide, except that one or more of the peptide bonds are replaced by an isostere pseudopeptide bond.
  • the most N-terminal peptide bond is substituted, since such a substitution would confer resistance to proteolysis by exopeptidases acting on the N-terminus.
  • the synthesis of peptides with one or more reduced isostere pseudopeptide bonds is known in the art (Couder, et al. (1993), cited above).
  • Other examples include the introduction of ketomethylene or methylsulfide bonds to replace peptide bonds.
  • Peptoid derivatives of peptides represent another class of peptide mimetics that retain the important structural determinants for biological activity, yet eliminate the peptide bonds, thereby conferring resistance to proteolysis (Simon, et al., 1992, Proc. Natl. Acad. Sci. USA, 89:9367-9371, incorporated herein by reference in its entirety).
  • Peptoids are oligomers of N-substituted glycines. A number of N-alkyl groups have been described, each corresponding to the side chain of a natural amino acid (Simon, et al. (1992), cited above). Some or all of the amino acids of the peptides may be replaced with the N-substituted glycine corresponding to the replaced amino acid.
  • peptide mimetic or “mimetic” also includes reverse-D peptides and enantiomers as defined below.
  • reverse-D peptide refers to a biologically active protein or peptide consisting of D-amino acids arranged in a reverse order as compared to the L-amino acid sequence of an peptide.
  • the carboxy terminal residue of an L-amino acid peptide becomes the amino terminal for the D-amino acid peptide and so forth.
  • ETESH SEQ ID NO: 117
  • H d S d E d T d E d the peptide, ETESH (SEQ ID NO: 117) becomes H d S d E d T d E d
  • E d , H d , S d , and T d are the D-amino acids corresponding to the L-amino acids, E, H, S, and T respectively.
  • enantiomer refers to a biologically active protein or peptide where one or more the L-amino acid residues in the amino acid sequence of an peptide is replaced with the corresponding D-amino acid residue(s).
  • composition refers broadly to any composition containing a recited peptide or amino acid sequence and, optionally an additional active agent.
  • the composition may comprise a dry formulation, an aqueous solution, or a sterile composition.
  • Compositions comprising peptides may be employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
  • the probe may be deployed in an aqueous solution containing salts, e.g., NaCl, detergents, e.g., sodium dodecyl sulfate (SDS), and other components, e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
  • salts e.g., NaCl
  • detergents e.g., sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • AUR acute urinary retention denotes a condition in which a mammal experiences a sudden inability to pass urine. It typically is painful and requires emergency treatment with a urinary catheter.
  • AUR can be caused by BPH, meatal stenosis, paraphimosis, penile constricting bands, phimosis, and prostate cancer.
  • AUR can be cause by prolapse (cystocele, rectocele, uterine), pelvic mass (gynaecological malignancy, uterine fibroid, ovarian cyst), or retroverted gravid uterus.
  • AUR can be caused by bladder calculi, bladder cancer, faecal impaction, gastrointestinal or retroperitoneal malignancy, urethral strictures, foreign bodies, and stones.
  • AUR is a complication of BPH and the reason for surgery in 20 to 30% of men undergoing prostatectomy. It may be spontaneous or it may also be precipitated by a number of factors in men with BPH such as anesthesia, medications, and unrelated surgery.
  • active agent is used to denote any agent capable of removing unwanted cellular proliferations and/or tissue growth.
  • suitable active agents may include, but are not limited to: (i) anti-cancer active agents (such as alkylating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, RNA/DNA antimetabolites, and antimitotic agents); (ii) active agents for treating benign growths such as anti-acne and anti-wart active agents; (iii) antiandrogen compounds, (cyproterone acetate (1 ⁇ , 2 ⁇ -methylene-6-chloro-17 ⁇ -acetoxy-6-dehydroprogesterone) Tamoxifen, aromatase inhibitors); (iv) alpha1-adrenergic receptor blockers (tamsulosin, terazosin, doxazosin, prazosin, bunazosin, indoramin,
  • the embodiments are directed to methods of preventing or reducing the incidence of acute urinary retention comprising administering to a mammal in need thereof a therapeutically effective amount of a composition comprising an NTP peptide, either alone or in combination with at least one additional active agent capable of treating and/or killing unwanted cellular proliferations in mammals.
  • the mammals treated are those that or are susceptible to having BPH.
  • the mammals treated are those that had been diagnosed with BPH and are currently being treated with alpha blockers such as tamsulosin, terazosin, doxazosin, or 5-alpha reductase inhibitors such as finasteride, dutasteride, or phosphodiesterase type 5 inhibitors (PDE5 inhibitors) such as tadalafil.
  • alpha blockers such as tamsulosin, terazosin, doxazosin, or 5-alpha reductase inhibitors such as finasteride, dutasteride, or phosphodiesterase type 5 inhibitors (PDE5 inhibitors) such as tadalafil.
  • PDE5 inhibitors phosphodiesterase type 5 inhibitors
  • NTP peptide sequences derived from an NTP peptide found to be an effective agent for causing cell death also may be used as an additional active agent in combination with the NTP peptides described herein.
  • a person ordinarily skilled in the art can, using the guidelines provided herein, synthesize without undue experimentation fragments of an effective Peptide spanning the entire amino acid sequence of that protein in order to identify other effective peptide sequences.
  • the inventor unexpectedly discovered that use of the NTP peptides in treating mammals in need of removal or destruction of unwanted cellular elements provided an unexpectedly superior reduction in the incidence of acute urinary retention.
  • the inventor unexpectedly discovered when conducting clinical trials for treating BPH, that the administration of the NTP peptides, alone or in combination with another active agent, dramatically reduced the incidence of acute urinary retention when compared to controls, and when compared to mammals that did not receive the NTP peptides, but also received active agents selected from alpha blockers such as tamsulosin, terazosin, doxazosin, or 5-alpha reductase inhibitors such as finasteride, dutasteride, or phosphodiesterase type 5 inhibitors (PDE5 inhibitors) such as tadalafil.
  • alpha blockers such as tamsulosin, terazosin, doxazosin, or 5-alpha reductase inhibitors
  • the inventor discovered during clinical trials that the incidence of acute urinary retention in men suffering from BPH, when treated with the claimed composition, alone or in combination with another active, was from about 0% to about 3%, or from about 0% to about 2.5%, or from about 0% to about 2.1%, or from about 0% to about 1.7%, or from about 0% to about 1%, or any value or range in between 0% and 3%.
  • compositions of the present invention exhibited a dramatic decrease in the incidence of acute urinary retention.
  • the method of the embodiments can prevent the incidence of acute urinary retention (incidence is 0%), or reduce the incidence rate by more than 10%, when compared to the controls.
  • administration of the compositions described herein, followed by administration of an additional active such as conventional alpha blockers such as tamsulosin, terazosin, doxazosin, or 5-alpha reductase inhibitors such as finasteride, dutasteride, or phosphodiesterase type 5 inhibitors (PDE5 inhibitors)
  • an additional active such as conventional alpha blockers such as tamsulosin, terazosin, doxazosin, or 5-alpha reductase inhibitors
  • finasteride, dutasteride, or phosphodiesterase type 5 inhibitors (PDE5 inhibitors) dramatically decreased in the incidence of acute urinary retention when compared to controls who received a control composition, followed by administration of
  • the decrease in acute urinary retention can be from about 10% to complete prevention (100%), or from about 25% to 100%, or from about 40% to 100%, or from about 40% to about 95%, or from about 45% to about 90%, or from about 50% to about 80%, from about 60% to about 75%, or any value or range between about 10% and about 100%.
  • the inventor also unexpectedly discovered when conducting clinical trials for treating an unrelated disorder, BPH, that the administration of the NTP peptides, alone or in combination with another active agent, dramatically reduced and/or prevented the incidence of acute urinary retention in patients at risk of developing acute urinary retention.
  • a patient at risk of developing acute urinary retention is a patient having can be a patient suffering from BPH, a patient suffering from BPH who is being treated with conventional alpha blockers such as tamsulosin, terazosin, doxazosin, or 5-alpha reductase inhibitors such as finasteride, dutasteride, or phosphodiesterase type 5 inhibitors (PDE5 inhibitors), a patient having acute urinary prostatitius, a mammal taking medications such as anticholinergic agents, tricyclic antidepressents, oral decongestants, nonsteroidal anti-inflammatory drugs, or any mammal having one or more of the conditions described in the tables below:
  • conventional alpha blockers such as tamsulosin, terazosin, doxazosin, or 5-alpha reductase inhibitors such as finasteride, dutasteride, or phosphodiesterase type 5 inhibitors (PDE5 inhibitors)
  • PDE5 inhibitors
  • compositions of the embodiments resulted in a reduction in the incidence of acute urinary retention to from about 0% to about 3%, or from about 0% to about 2.5%, or from about 0% to about 2.1%, or from about 0% to about 1.7%, or from about 0% to about 1%, or any value or range in between 0% and 3%.
  • compositions of the embodiments resulted in a reduction in the incidence of acute urinary retention, when compared to similarly-treated patients who are not administered a composition of the embodiments, of from about 10% to complete prevention (100%), or from about 25% to 100%, or from about 40% to 100%, or from about 40% to about 95%, or from about 45% to about 90%, or from about 50% to about 80%, from about 60% to about 75%, or any value or range between about 10% and about 100%.
  • the embodiments include a method of treating a mammal suffering from a condition that may lead to acute urinary retention, comprising administering once or more than once an NTP peptide to the mammal, either alone or in combination with administration of an additional active agent.
  • the method includes, but is not limited to, administering the NTP-peptides intramuscularly, orally, intravenously, intraperitoneally, intracerebrally (intraparenchymally), intracerebroyentricularly, intralesionally, intraocularly, intraarterially, intrathecally, intratumorally, intranasally, topically, transdermally, subcutaneously, or intradermally, either alone or conjugated to a carrier.
  • the condition that may lead to acute urinary retention may include the presence of unwanted cellular proliferations including, inter alia, benign and malignant tumors, glandular (e.g. prostate) hyperplasia, and cancer.
  • Preferred NTP peptides include one or more of the following:
  • Any mammal can benefit from use of the invention, including humans, mice, rabbits, dogs, sheep and other livestock, any mammal treated or treatable by a veterinarian, zoo-keeper, or wildlife preserve employee.
  • Preferred mammals are humans, sheep, and dogs. Throughout this description mammals and patients are used interchangeably.
  • the peptides of the embodiments encompass these other fragments.
  • the peptides of the embodiments have at least 4 amino acids, preferably at least 5 amino acids, and more preferably at least 6 amino acids.
  • the embodiments also encompass methods of preventing or reducing the incidence of acute urinary retention comprising administering a composition comprising NTP peptides comprising two or more NTP peptides joined together, together with an additional active agent.
  • a composition comprising NTP peptides comprising two or more NTP peptides joined together, together with an additional active agent.
  • an NTP peptide has the desired biological activity, it follows that two such Peptides would also possess the desired biological activity.
  • NTP peptides and fragments, variants, derivatives, homologues, fusion proteins and mimetics thereof encompassed by this embodiment can be prepared using methods known to those of skill in the art, such as recombinant DNA technology, protein synthesis and isolation of naturally occurring peptides, proteins, AD7c-protein and fragments, variants, derivatives and homologues thereof.
  • NTP peptides and fragments, variants, derivatives, homologues, fusion proteins and mimetics thereof can be prepared from other peptides, proteins, and fragments, variants, derivatives and homologues thereof using methods known to those having skill in the art. Such methods include (but are not limited to) the use of proteases to cleave the peptide, or protein into the desired NTP peptides.
  • NTP peptide can be prepared using well known recombinant DNA technology methods such as those set forth in Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and/or Ausubel et al., eds., Current Protocols in Molecular Biology, Green Publishers Inc. and Wiley and Sons, N.Y.
  • a gene or cDNA encoding an NTP peptide may be obtained for example by screening a genomic or cDNA library, or by PCR amplification. Probes or primers useful for screening the library can be generated based on sequence information for other known genes or gene fragments from the same or a related family of genes, such as, for example, conserved motifs found in other peptides or proteins. In addition, where a gene encoding an NTP peptide has been identified, all or a portion of that gene may be used as a probe to identify homologous genes. The probes or primers may be used to screen cDNA libraries from various tissue sources believed to express an NTP peptide gene. Typically, conditions of high stringency will be employed for screening to minimize the number of false positives obtained from the screen.
  • Another means to prepare a gene encoding an NTP peptide is to employ chemical synthesis using methods well known to the skilled artisan, such as those described by Engels et al., Angew. Chem. Intl. Ed., 28:716-734. These methods include, inter alia, the phosphotriester, phosphoramidite, and H-phosphonate methods for nucleic acid synthesis. A preferred method for such chemical synthesis is polymer-supported synthesis using standard phosphoramidite chemistry.
  • the DNA encoding an peptide or protein will be several hundred nucleotides in length. Nucleic acids larger than about 100 to nucleotides can be synthesized as several fragments using these methods.
  • the fragments then can be ligated together to form the full length peptide or protein.
  • the DNA fragment encoding the amino terminus of the protein will have an ATG, which encodes a methionine residue. This methionine may or may not be present on the mature form of the protein or peptide, depending on whether the protein produced in the host cell is designed to be secreted from that cell.
  • the gene, cDNA, or fragment thereof encoding the NTP peptide can be inserted into an appropriate expression or amplification vector using standard ligation techniques.
  • the vector is typically selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery such that amplification of the gene and/or expression of the gene can occur).
  • the gene, cDNA or fragment thereof encoding the NTP peptide may be amplified/expressed in prokaryotic, yeast, insect (baculovirus systems) and/or eukaryotic host cells. Selection of the host cell will depend in part on whether the NTP peptide is to be glycosylated and/or phosphorylated. If so, yeast, insect, or mammalian host cells are preferable.
  • the vectors used in any of the host cells will contain at least a 5′ flanking sequence (also referred to as a promoter) and other regulatory elements as well, such as an enhancer(s), an origin of replication element, a transcriptional termination element, a complete intron sequence containing a donor and acceptor splice site, a signal peptide sequence, a ribosome binding site element, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.
  • a 5′ flanking sequence also referred to as a promoter
  • other regulatory elements such as an enhancer(s), an origin of replication element, a transcriptional termination element, a complete intron sequence containing a donor and acceptor splice site, a signal peptide sequence, a ribosome binding site element, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be
  • the vector may contain a tag sequence, i.e., an oligonucleotide molecule located at the 5′ or 3′ end of the protein or peptide coding sequence; the oligonucleotide molecule encodes polyHis (such as hexaHis (SEQ ID NO: 118)), or other tag such as FLAG, HA (hemaglutinin Influenza virus) or myc for which commercially available antibodies exist.
  • This tag is typically fused to the polypeptide upon expression of the polypeptide, and can serve as means for affinity purification of the protein or peptide from the host cell. Affinity purification can be accomplished, for example, by column chromatography using antibodies against the tag as an affinity matrix.
  • the tag can subsequently be removed from the purified protein or peptide by various means such as using certain peptidases.
  • the human immunoglobulin hinge and Fc region could be fused at either the N-terminus or C-terminus of the NTP peptide by one skilled in the art.
  • the subsequent Fc-fusion protein could be purified by use of a Protein A affinity column.
  • Fc is known to exhibit a long pharmacokinetic half-life in vivo and proteins fused to Fc have been found to exhibit a substantially greater half-life in vivo than the unfused counterpart. Also, fusion to the Fc region allows for dimerization/multimerization of the molecule that may be useful for the bioactivity of some molecules.
  • the 5′ flanking sequence may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of 5′ flanking sequences from more than one source), synthetic, or it may be the native protein or peptide gene 5′ flanking sequence.
  • the source of the 5′ flanking sequence may be any unicellular prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the 5′ flanking sequence is functional in, and can be activated by, the host cell machinery.
  • the 5′ flanking sequences useful in the vectors of this embodiment may be obtained by any of several methods well known in the art. Typically, 5′ flanking sequences useful herein other than the protein or peptide gene flanking sequence will have been previously identified by mapping and/or by restriction endonuclease digestion and can thus be isolated from the proper tissue source using the appropriate restriction endonucleases. In some cases, the full nucleotide sequence of the 5′ flanking sequence may be known. Here, the 5′ flanking sequence may be synthesized using the methods described above for nucleic acid synthesis or cloning. Where all or only a portion of the 5′ flanking sequence is known, it may be obtained using PCR and/or by screening a genomic library with suitable oligonucleotide and/or 5′ flanking sequence fragments from the same or another species.
  • a fragment of DNA containing a 5′ flanking sequence may be isolated from a larger piece of DNA that may contain, for example, a coding sequence or even another gene or genes. Isolation may be accomplished by restriction endonuclease digestion using one or more carefully selected enzymes to isolate the proper DNA fragment. After digestion, the desired fragment may be isolated by agarose gel purification, Qiagen® column or other methods known to the skilled artisan. Selection of suitable enzymes to accomplish this purpose will be readily apparent to one of ordinary skill in the art.
  • the origin of replication element is typically a part of prokaryotic expression vectors purchased commercially, and aids in the amplification of the vector in a host cell. Amplification of the vector to a certain copy number can, in some cases, be important for optimal expression of the protein or peptide. If the vector of choice does not contain an origin of replication site, one may be chemically synthesized based on a known sequence, and ligated into the vector.
  • the transcription termination element is typically located 3′ of the end of the protein or peptide coding sequence and serves to terminate transcription of the protein or peptide. Usually, the transcription termination element in prokaryotic cells is a G-C rich fragment followed by a poly T sequence. While the element may be cloned from a library or purchased commercially as part of a vector, it can also be readily synthesized using methods for nucleic acid synthesis such as those described above.
  • a selectable marker gene element encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium.
  • Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells, (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex media.
  • Preferred selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene.
  • the ribosome binding element commonly called the Shine-Dalgarno sequence (prokaryotes) or the Kozak sequence (eukaryotes), is usually necessary for translation initiation of mRNA.
  • the element is typically located 3′ to the promoter and 5′ to the coding sequence of the protein or peptide to be synthesized.
  • the Shine-Dalgarno sequence is varied but is typically a polypurine (i.e., having a high A-G content). Many Shine-Dalgarno sequences have been identified, each of which can be readily synthesized using methods set forth above and used in a prokaryotic vector.
  • a signal sequence may be used to direct the Peptide out of the host cell where it is synthesized, and the carboxy-terminal part of the protein may be deleted in order to prevent membrane anchoring.
  • the signal sequence is positioned in the coding region of the NTP peptide gene or cDNA, or directly at the 5′ end of the Peptide gene coding region.
  • Many signal sequences have been identified, and any of them that are functional in the selected host cell may be used in conjunction with the Peptide gene or cDNA. Therefore, the signal sequence may be homologous or heterologous to the Peptide gene or cDNA, and may be homologous or heterologous to the Peptide gene or cDNA.
  • the signal sequence may be chemically synthesized using methods set forth above. In most cases, secretion of the polypeptide from the host cell via the presence of a signal peptide will result in the removal of the amino terminal methionine from the polypeptide.
  • transcription of the NTP peptide gene or cDNA is increased by the presence of one or more introns in the vector; this is particularly true where the Peptide is produced in eukaryotic host cells, especially mammalian host cells.
  • the introns used may be naturally occurring within the Peptide gene, especially where the gene used is a full length genomic sequence or a fragment thereof. Where the intron is not naturally occurring within the gene (as for most cDNAs), the intron(s) may be obtained from another source.
  • the position of the intron with respect to the flanking sequence and the Peptide gene generally is important, as the intron must be transcribed to be effective.
  • the preferred position for the intron is 3′ to the transcription start site, and 5′ to the polyA transcription termination sequence.
  • the intron or introns will be located on one side or the other (i.e., 5′ or 3′) of the cDNA such that it does not interrupt this coding sequence.
  • Any intron from any source including any viral, prokaryotic and eukaryotic (plant or animal) organisms, may be used to practice this embodiment, provided that it is compatible with the host cell(s) into which it is inserted.
  • synthetic introns may be used in the vector.
  • the final vectors used to practice this embodiment may be constructed from starting vectors such as a commercially available vector. Such vectors may or may not contain some of the elements to be included in the completed vector. If none of the desired elements are present in the starting vector, each element may be individually ligated into the vector by cutting the vector with the appropriate restriction endonuclease(s) such that the ends of the element to be ligated in and the ends of the vector are compatible for ligation. In some cases, it may be necessary to blunt the ends to be ligated together in order to obtain a satisfactory ligation. Blunting is accomplished by first filling in “sticky ends” using Klenow DNA polymerase or T4 DNA polymerase in the presence of all four nucleotides.
  • two or more of the elements to be inserted into the vector may first be ligated together (if they are to be positioned adjacent to each other) and then ligated into the vector.
  • An additional method for constructing the vector is to conduct all ligations of the various elements simultaneously in one reaction mixture.
  • many nonsense or nonfunctional vectors will be generated due to improper ligation or insertion of the elements, however the functional vector may be identified and selected by restriction endonuclease digestion.
  • Preferred vectors for practicing this embodiment are those that are compatible with bacterial, insect, and mammalian host cells.
  • Such vectors include, inter alia, pCRII, pCR3, and pcDNA3.1 (Invitrogen Company, San Diego, Calif.), pBSII (Stratagene Company, La Jolla, Calif.), pET15b (Novagen, Madison, Wis.), PGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL (BlueBachl; Invitrogen), and pFastBacDual (Gibco/BRL, Grand Island, N.Y.).
  • the completed vector may be inserted into a suitable host cell for amplification and/or polypeptide expression.
  • Host cells may be prokaryotic host cells (such as E. coli ) or eukaryotic host cells (such as a yeast cell, an insect cell, or a vertebrate cell).
  • the host cell when cultured under appropriate conditions, can synthesize protein or peptide which can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted).
  • the NTP peptide can be purified using methods such as molecular sieve chromatography, affinity chromatography, and the like. Selection of the host cell for protein or peptide production will depend in part on whether the Peptide is to be glycosylated or phosphorylated (in which case eukaryotic host cells are preferred), and the manner in which the host cell is able to fold the Peptide into its native tertiary structure (e.g., proper orientation of disulfide bridges, etc.) such that biologically active protein is prepared by the Peptide that has biological activity, the Peptide may be folded after synthesis using appropriate chemical conditions as discussed below.
  • Suitable cells or cell lines may be mammalian cells, such as Chinese hamster ovary cells (CHO), human embryonic kidney (HEK) 293, 293T cells, or 3T3 cells.
  • CHO Chinese hamster ovary cells
  • HEK human embryonic kidney
  • 3T3 cells 3T3 cells.
  • the selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art.
  • Other suitable mammalian cell lines are the monkey COS-1 and COS-7 cell lines, and the CV-1 cell line.
  • Further exemplary mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants, are also suitable.
  • Candidate cells may be genotypically deficient in the selection gene, or may contain a dominantly acting selection gene.
  • suitable mammalian cell lines include but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines.
  • E. coli e.g., HB101, DH5.alpha., DH10, and MC1061
  • B. subtilis Various strains of B. subtilis, Pseudomonas spp., other Bacillus spp., Streptomyces spp., and the like may also be employed in this method.
  • Many strains of yeast cells known to those skilled in the art also are available as host cells for expression of the polypeptides of the present embodiments.
  • insect cell systems may be utilized in the methods of the present embodiments. Such systems are described for example in Kitts et al. (Biotechniques, 14:810-817), Lucklow (Curr. Opin. Biotechnol., 4:564-572) and Lucklow et al. (J. Virol., 67:4566-4579).
  • Preferred insect cells are Sf-9 and Hi5 (Invitrogen, Carlsbad, Calif.).
  • Insertion also referred to as transformation or transfection
  • transformation or transfection of the vector into the selected host cell may be accomplished using such methods as calcium chloride, electroporation, microinjection, lipofection, or the DEAE-dextran method.
  • the method selected will in part be a function of the type of host cell to be used.
  • the host cells containing the vector may be cultured using standard media well known to the skilled artisan.
  • the media will usually contain all nutrients necessary for the growth and survival of the cells.
  • Suitable media for culturing E. coli cells are for example, Luria Broth (LB) and/or Terrific Broth (TB).
  • Suitable media for culturing eukaryotic cells are RPMI 1640, MEM, DMEM, all of which may be supplemented with serum and/or growth factors as required by the particular cell line being cultured.
  • a suitable medium for insect cultures is Grace's medium supplemented with yeastolate, lactalbumin hydrolysate, and/or fetal calf serum as necessary.
  • an antibiotic or other compound useful for selective growth of the transformed cells is added as a supplement to the media.
  • the compound to be used will be dictated by the selectable marker element present on the plasmid with which the host cell was transformed.
  • the selectable marker element is kanamycin resistance
  • the compound added to the culture medium will be kanamycin.
  • the amount of NTP peptide produced in the host cell can be evaluated using standard methods known in the art. Such methods include, without limitation, Western blot analysis, SDS-polyacrylamide gel electrophoresis, non-denaturing gel electrophoresis, HPLC separation, mass spectroscopy, immunoprecipitation, and/or activity assays such as DNA binding gel shift assays.
  • the protein or peptide has been designed to be secreted from the host cells, the majority of the protein or peptide may be found in the cell culture medium. Proteins prepared in this way will typically not possess an amino terminal methionine, as it is removed during secretion from the cell. If however, the protein or peptide is not secreted from the host cells, it will be present in the cytoplasm and/or the nucleus (for eukaryotic host cells) or in the cytosol (for gram negative bacteria host cells) and may have an amino terminal methionine.
  • the host cells are typically first disrupted mechanically or with detergent to release the intra-cellular contents into a buffered solution.
  • the Peptide can then be isolated from this solution.
  • NTP peptides from solution can be accomplished using a variety of techniques. If the NTP peptide has been synthesized such that it contains a tag such as hexaHistidine (SEQ ID NO: 118) (e.g. peptide/hexaHis (SEQ ID NO: 118)) or other small peptide such as FLAG (Sigma-Aldritch, St.
  • a tag such as hexaHistidine (SEQ ID NO: 118) (e.g. peptide/hexaHis (SEQ ID NO: 118)) or other small peptide such as FLAG (Sigma-Aldritch, St.
  • calmodulin-binding peptide (Stratagene, La Jolla, Calif.) at either its carboxyl or amino terminus, it may essentially be purified in a one-step process by passing the solution through an affinity column where the column matrix has a high affinity for the tag or for the protein directly (i.e., a monoclonal antibody specifically recognizing the peptide).
  • polyhistidine binds with great affinity and specificity to nickel, zinc and cobalt; thus immobilized metal ion affinity chromatography which employs a nickel-based affinity resin (as used in Qiagen's QIAexpress system or Invitrogen's Xpress System) or a cobalt-based affinity resin (as used in BD Biosciences-CLONTECH's Talon system) can be used for purification of peptide/polyHis.
  • a nickel-based affinity resin as used in Qiagen's QIAexpress system or Invitrogen's Xpress System
  • cobalt-based affinity resin as used in BD Biosciences-CLONTECH's Talon system
  • NTP peptide is prepared without a tag attached, and no antibodies are available
  • other well known procedures for purification include, without limitation, ion exchange chromatography, hydroxyapatite chromatography, hydrophobic interaction chromatography, molecular sieve chromatography, HPLC, native gel electrophoresis in combination with gel elution, and preparative isoelectric focusing (Isoprime machine/technique, Hoefer Scientific). In some cases, two or more of these techniques may be combined to achieve increased purity.
  • the intracellular material can be extracted from the host cell using any standard technique known to the skilled artisan.
  • the host cells can be lysed to release the contents of the periplasm/cytoplasm by French press, homogenization, and/or sonication followed by centrifugation. If the Peptide has formed inclusion bodies in the cytosol, the inclusion bodies can often bind to the inner and/or outer cellular membranes and thus will be found primarily in the pellet material after centrifugation.
  • the pellet material then can be treated at pH extremes or with chaotropic agent such as a detergent, guanidine, guanidine derivatives, urea, or urea derivatives in the presence of a reducing agent such as dithiothreitol at alkaline pH or tris carboxyethyl phosphine at acid pH to release, break apart, and solubilize the inclusion bodies.
  • chaotropic agent such as a detergent, guanidine, guanidine derivatives, urea, or urea derivatives in the presence of a reducing agent such as dithiothreitol at alkaline pH or tris carboxyethyl phosphine at acid pH to release, break apart, and solubilize the inclusion bodies.
  • a reducing agent such as dithiothreitol at alkaline pH or tris carboxyethyl phosphine at acid pH to release, break apart, and solubilize the inclusion bodies.
  • the NTP peptide may not be biologically active upon isolation.
  • Various methods for refolding or converting the polypeptide to its tertiary structure and generating disulfide linkages can be used to restore biological activity. Such methods include exposing the solubilized polypeptide to a pH usually above 7 and in the presence of a particular concentration of a chaotrope. The selection of chaotrope is very similar to the choices used for inclusion body solubilization but usually at a lower concentration and is not necessarily the same chaotrope as used for the solubilization.
  • the refolding/oxidation solution will also contain a reducing agent or the reducing agent plus its, oxidized form in a specific ratio to generate a particular redox potential allowing for disulfide shuffling to occur in the formation of the protein's cysteine bridge(s).
  • Some of the commonly used redox couples include cysteine/cystamine, glutathione (GSH)/dithiobis GSH, cupric chloride, dithiothreitol (DTT)/dithiane DTT, 2-mercaptoethanol (bME)/dithio-b (ME).
  • a cosolvent is necessary to increase the efficiency of the refolding and the more common reagents used for this purpose include glycerol, polyethylene glycol of various molecular weights, and arginine.
  • NTP peptide inclusion bodies are not formed to a significant degree in the host cell, the NTP peptide will be found primarily in the supernatant after centrifugation of the cell homogenate, and the NTP peptide can be isolated from the supernatant using methods such as those set forth below.
  • purification can be accomplished using standard methods well known to the skilled artisan. Such methods include, without limitation, separation by electrophoresis followed by electroelution, various types of chromatography (immunoaffinity, molecular sieve, and/or ion exchange), and/or high pressure liquid chromatography. In some cases, it may be preferable to use more than one of these methods for complete purification.
  • NTP peptides and their fragments, variants, homologues, fusion proteins, peptide mimetics, and derivatives may be prepared by chemical synthesis methods (such as solid phase peptide synthesis) using techniques known in the art such as those set forth by Merrifield et al., J. Am. Chem. Soc., 85:2149, Houghten et al. Proc Natl Acad. Sci. USA, 82:5132, and Stewart and Young, Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, Ill. Such Peptides may be synthesized with or without a methionine on the amino terminus.
  • NTP peptides may be oxidized using methods set forth in these references to form disulfide bridges.
  • the NTP peptides are expected to have biological activity comparable to Peptides produced recombinantly or purified from natural sources, and thus may be used interchangeably with recombinant or natural Peptide.
  • Chemically modified NTP peptide compositions in which the Peptide is linked to a polymer are included within the scope of the present embodiments.
  • the polymer selected is typically water soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment.
  • the polymer selected is usually modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization may be controlled as provided for in the present methods.
  • the polymer may be of any molecular weight, and may be branched or unbranched. Included within the scope of peptide polymers is a mixture of polymers.
  • nucleic acid and/or amino acid variants of the naturally occurring NTP peptides may be produced using site directed mutagenesis, PCR amplification, or other appropriate methods, where the primer(s) have the desired point mutations (see Sambrook et al., supra, and Ausubel et al., supra, for descriptions of mutagenesis techniques). Chemical synthesis using methods described by Engels et al., supra, may also be used to prepare such variants. Other methods known to the skilled artisan may be used as well.
  • Preferred nucleic acid variants are those containing nucleotide substitutions accounting for codon preference in the host cell that is to be used to produce NTP peptides.
  • Such codon optimization can be determined via computer algorithers which incorporate codon frequency tables such as Ecohigh. Cod for codon preference of highly expressed bacterial genes as provided by the University of Wisconsin Package Version 9.0, Genetics Computer Group, Madison, Wis.
  • Other useful codon frequency tables include Celegans_high.cod, Celegans_low.cod, Drosophila _high.cod, Human_high.cod, Maize_high.cod, and Yeast_high.cod.
  • Other preferred variants are those encoding conservative amino acid changes as described above (e.g., wherein the charge or polarity of the naturally occurring amino acid side chain is not altered substantially by substitution with a different amino acid) as compared to wild type, and/or those designed to either generate a novel glycosylation and/or phosphorylation site(s), or those designed to delete an existing glycosylation and/or phosphorylation site(s).
  • NTP peptides and fragments, homologs, variants, fusion proteins, peptide mimetics, derivatives and salts thereof also can be made using conventional peptide synthesis techniques known to the skilled artisan. These techniques include chemical coupling methods (cf. Wunsch, E: “Methoden der organischen Chemie”, Volume 15, Band 1+2, Synthese von Peptiden, thime Verlag, Stuttgart (1974), and Barrany, G.; Merrifield, R. B.: “The Peptides,” eds. E. Gross, J. Meienhofer, Volume 2, Chapter 1, pp. 1-284, Academic Press (1980)), enzymatic coupling methods (cf. Widmer, F. Johansen, J. T., Carlsberg Res.
  • peptide mimetics are more bioavailable, have a longer duration of action and can be cheaper to produce than the native proteins and peptides.
  • Peptide mimetics of NTP peptides can be developed using combinatorial chemistry techniques and other techniques known in the art (see e.g. Proceedings of the 20th European Peptide Symposium, ed. G. Jung, E. Bayer, pp. 289-336, and references therein).
  • Examples of methods known in the art for structurally modifying a peptide to create a peptide mimetic include the inversion of backbone chiral centers leading to D-amino acid residue structures that may, particularly at the N-terminus, lead to enhanced stability for proteolytical degradation without adversely affecting activity.
  • An example is provided in the paper “Tritriated D-ala.sup.1-Peptide T Binding”, Smith C. S. et al., Drug Development Res. 15, pp. 371-379 (1988).
  • a second method is altering cyclic structure for stability, such as N to C interchain imides and lactames (Ede et al. in Smith and Rivier (Eds.) “Peptides: Chemistry and Biology”, Escom, Leiden (1991), pp. 268-270).
  • An example of this is given in conformationally restricted thymopentin-like compounds, such as those disclosed in U.S. Pat. No. 4,457,489 (1985), Goldstein, G. et al., the disclosure of which is incorporated by reference herein in its entirety.
  • a third method is to substitute peptide bonds in the NTP peptide by pseudopeptide bonds that confer resistance to proteolysis.
  • pseudopeptide bonds have been described that in general do not affect peptide structure and biological activity.
  • One example of this approach is to substitute retro-inverso pseudopeptide bonds (“Biologically active retroinverso analogues of thymopentin”, Sisto A. et al in Rivier, J. E. and Marshall, G. R. (eds) “Peptides, Chemistry, Structure and Biology”, Escom, Leiden (1990), pp. 722-773) and Dalpozzo, et al. (1993), Int. J. Peptide Protein Res., 41:561-566, incorporated herein by reference).
  • the amino acid sequences of the peptides may be identical to the sequences of the peptides described above, except that one or more of the peptide bonds are replaced by a retro-inverso pseudopeptide bond.
  • the most N-terminal peptide bond is substituted, since such a substitution will confer resistance to proteolysis by exopeptidases acting on the N-terminus.
  • peptide bonds can be replaced by non-peptide bonds that allow the peptide mimetic to adopt a similar structure, and therefore biological activity, to the original peptide. Further modifications also can be made by replacing chemical groups of the amino acids with other chemical groups of similar structure.
  • Another suitable pseudopeptide bond that is known to enhance stability to enzymatic cleavage with no or little loss of biological activity is the reduced isostere pseudopeptide bond is a (Couder, et al. (1993), Int. J.
  • amino acid sequences of these peptides may be identical to the sequences of an peptide, except that one or more of the peptide bonds are replaced by an isostere pseudopeptide bond.
  • amino acid sequences of these peptides may be identical to the sequences of an peptide, except that one or more of the peptide bonds are replaced by an isostere pseudopeptide bond.
  • the most N-terminal peptide bond is substituted, since such a substitution would confer resistance to proteolysis by exopeptidases acting on the N-terminus.
  • the synthesis of peptides with one or more reduced isostere pseudopeptide bonds is known in the art (Couder, et al. (1993), cited above).
  • Other examples include the introduction of ketomethylene or methylsulfide bonds to replace peptide bonds.
  • NTP peptides represent another class of peptide mimetics that retain the important structural determinants for biological activity, yet eliminate the peptide bonds, thereby conferring resistance to proteolysis (Simon, et al., 1992, Proc. Natl. Acad. Sci. USA, 89:9367-9371 and incorporated herein by reference in its entirety).
  • Peptoids are oligomers of N-substituted glycines. A number of N-alkyl groups have been described, each corresponding to the side chain of a natural amino acid (Simon, et al. (1992), cited above and incorporated herein by reference in its entirety). Some or all of the amino acids of the peptide are replaced with the N-substituted glycine corresponding to the replaced amino acid.
  • peptide mimetics can be aided by determining the tertiary structure of the original peptide by NMR spectroscopy, crystallography and/or computer-aided molecular modeling. These techniques aid in the development of novel compositions of higher potency and/or greater bioavailability and/or greater stability than the original peptide (Dean (1994), BioEssays, 16: 683-687; Cohen and Shatzmiller (1993), J. Mol. Graph., 11: 166-173; Wiley and Rich (1993), Med. Res. Rev., 13: 327-384; Moore (1994), Trends Pharmacol. Sci., 15: 124-129; Hruby (1993), Biopolymers, 33: 1073-1082; Bugg et al. (1993), Sci. Am., 269: 92-98, all incorporated herein by reference in their entirety).
  • a potential peptide mimetic compound may be synthesized and assayed using the methods outlined in the examples below to assess its activity.
  • the peptide mimetic compounds obtained by the above methods having the biological activity of the peptides and similar three-dimensional structure, are encompassed by this embodiment. It will be readily apparent to one skilled in the art that a peptide mimetic can be generated from any of the peptides bearing one or more of the modifications described above. It will furthermore be apparent that the peptide mimetics of this embodiment can be further used for the development of even more potent non-peptidic compounds, in addition to their utility as therapeutic compounds.
  • the present embodiments are directed to methods of treating mammals with conditions that may result in acute urinary retention, where the condition may be a condition requiring removal of cells, such as benign and malignant tumors, glandular (e.g. prostate) hyperplasia, or cancer.
  • the methods of treatment reduce the incidence of acute urinary retention, and some cases prevents acute urinary retention (i.e., and incidence of 0%).
  • Such methods comprise administering to a mammal in need thereof, a therapeutically effective amount of NTP peptide, either alone, or in combination with an additional active agent.
  • the mammals in need may be mammals suffering from benign prostatic hyperplasia, that may or may not be at increased risk of developing prostate cancer, or mammals at risk of developing prostate cancer.
  • the mammals in need also may be any mammal that would benefit from a reduction in the incidence of acute urinary retention such as mammals having one or more of the conditions listed in Tables 4-6 above.
  • the additional active agent can be one or more active agents selected from (i) anti-cancer active agents (such as alkylating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, RNA/DNA antimetabolites, and antimitotic agents); (ii) active agents for treating benign growths such as anti-acne and anti-wart active agents (salicylic acid); (iii) antiandrogen compounds, (cyproterone acetate (1 ⁇ , 2 ⁇ -methylene-6-chloro-17 ⁇ -acetoxy-6-dehydroprogesterone)) Tamoxifen, aromatase inhibitors); (iv) alpha1-adrenergic receptor blockers (tamsulosin, terazosin, doxazosin, prazosin, bunazosin, indoramin, alfulzosin, silodosin); (v) 5 ⁇ -reductase inhibitors (finasteride, du
  • the additional active agent is selected from the group consisting of tamsulosin, finasteride, terazosin, doxazosin, prazosin, tadalafil, alfuzosin, silodosin, dutasteride, combinations of dutasteride and tamsulosin, and mixtures and combinations thereof.
  • the condition can be, for example, tumors of lung, breast, stomach, pancreas, prostate, bladder, bone, ovary, skin, kidney, sinus, colon, intestine, stomach, rectum, esophagus, blood, brain and its coverings, spinal cord and its coverings, muscle, connective tissue, adrenal, parathyroid, thyroid, uterus, testis, pituitary, reproductive organs, liver, gall bladder, eye, ear, nose, throat, tonsils, mouth, lymph nodes and lymphoid system, and other organs.
  • Other conditions include stroke or congestive heart failure, a medical condition such as prostatitis or urinary tract infection, or ingestion of medication or drugs known to precipitate retention, e.g., pseudoephedrine hydrochloride, cold medicine, pain medication such as narcotics or sedatives, or benadryl, or any other conditions for either male or female mammals listed in Tables 4-6 above.
  • the condition is benign prostatic hyperplasia.
  • malignant tumor is intended to encompass all forms of human carcinomas, sarcomas and melanomas which occur in the poorly differentiated, moderately differentiated, and well-differentiated forms.
  • compositions of NTP peptides may comprise a therapeutically effective amount of an NTP peptide in admixture with a pharmaceutically acceptable carrier.
  • the additional active agent can be administered in the same composition with the NTP peptide, and in other embodiments, the composition comprising the NTP peptide is administered as an injection, whereas the additional active agent is formulated into an oral medication (gel, capsule, tablet, liquid, etc.).
  • the carrier material may be water for injection, preferably supplemented with other materials common in solutions for administration to mammals.
  • an NTP peptide for therapeutic use will be administered in the form of a composition comprising purified peptide in conjunction with one or more physiologically acceptable carriers, excipients, or diluents.
  • Neutral buffered saline or saline mixed with serum albumin are exemplary appropriate carriers.
  • the product is formulated as a lyophilizate using appropriate excipients (e.g., sucrose).
  • excipients e.g., sucrose
  • Other standard carriers, diluents, and excipients may be included as desired.
  • Compositions of the embodiments also may comprise buffers known to those having ordinary skill in the art with an appropriate range of pH values, including Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute therefor.
  • NTP peptides conjugated or linked or bound to an antibody, antibody fragment, antibody-like molecule, or a molecule with a high affinity to a specific tumor marker such as a cellular receptor, signal peptide or over-expressed enzyme
  • a specific tumor marker such as a cellular receptor, signal peptide or over-expressed enzyme
  • the antibody, antibody fragment, antibody-like molecule, or molecule with a high affinity to a specific tumor marker is used to target the Peptide conjugate to a specific cellular or tissue target.
  • a tumor with a distinctive surface antigen or expressed antigen can be targeted by the antibody, antibody fragment, or antibody-like binding molecule and the tumor cells can be killed by the Peptide.
  • Such an approach using antibody targeting has the anticipated advantages of decreasing dosage, increasing the likelihood of binding to and uptake by the target cells, and increased usefulness for targeting and treating metastatic tumors and microscopic sized tumors.
  • the embodiments also encompass the use of NTP peptides conjugated or linked or bound to a protein or other molecule to form a composition that, upon cleavage at or near the site(s) of the tumor or other unwanted cells by a tumor- or site-specific enzyme or protease or by an antibody conjugate that targets tumor or other unwanted cells, releases the Peptide at or near the site(s) of the tumor or other unwanted cells
  • the embodiments also encompass the use of NTP peptides conjugated or linked or bound to a protein or other molecule to form a composition that releases the Peptide or some biologically active fragment of the Peptide upon exposure of the tissue to be treated to light (as in laser therapies or other photo-dynamic or photo-activated therapy), other forms of electromagnetic radiation such as infra-red radiation, ultraviolet radiation, x-ray or gamma ray radiation, localized heat, alpha or beta radiation, ultrasonic emissions, or other sources of localized energy.
  • electromagnetic radiation such as infra-red radiation, ultraviolet radiation, x-ray or gamma ray radiation, localized heat, alpha or beta radiation, ultrasonic emissions, or other sources of localized energy.
  • the embodiments also encompass therapeutic compositions of NTP peptides employing dendrimers, fullerenes, and other synthetic molecules, polymers and macromolecules where the Peptide and/or its corresponding DNA molecule is conjugated with, attached to or enclosed in the molecule, polymer or macromolecule, either by itself or in conjunction with other species of molecule such as a tumor-specific marker.
  • U.S. Pat. No. 5,714,166 Bioactive and/or Targeted Dendimer Conjugates, provides a method of preparing and using, inter alia, dendritic polymer conjugates composed of at least one dendrimer with a target director(s) and at least one bioactive agent conjugated to it.
  • the disclosure of U.S. Pat. No. 5,714,166 is incorporated by reference herein in its entirety.
  • the embodiments also encompasses methods of treating mammals with therapeutic compositions of NTP peptides and/or genes and drug delivery vehicles such as lipid emulsions, micelle polymers, polymer microspheres, electroactive polymers, hydrogels and liposomes, in combination with an additional active agent.
  • therapeutic compositions of NTP peptides and/or genes and drug delivery vehicles such as lipid emulsions, micelle polymers, polymer microspheres, electroactive polymers, hydrogels and liposomes, in combination with an additional active agent.
  • NTP peptides or related genes or gene equivalents transferred to the unwanted cells in a mammal also is encompassed by the embodiments.
  • Overexpression of the NTP peptide within the tumor can be used to induce the cells in the tumor to die and thus reduce the tumor cell population.
  • the gene or gene equivalent transfer of NTP peptide to treat the unwanted cellular elements is anticipated to have the advantage of requiring less dosage, and of being passed on to the cellular progeny of the targeted cellular elements, thus necessitating less frequent therapy, and less total therapy.
  • This embodiment also encompasses the transfer of genes that code for a fusion protein containing an NTP peptide to the unwanted cells or neighboring cells where, following the expression of the gene and the production and/or secretion of the fusion protein, the fusion protein is cleaved either by native enzymes or proteases or by a prodrug to release the NTP peptide in, at or near the unwanted cells.
  • cloned recombinant peptide-antibody conjugates for administration to treatment na ⁇ ve mammals also is encompassed by the scope of the embodiments.
  • targeting conjugate such as an antibody, antibody fragment, antibody-like molecule, or a molecule with a high affinity to a cancer-specific receptor or other tumor marker
  • the embodiments further include the use of therapeutic compositions of NTP peptides or genes or gene equivalents as a component of the coating of a medical device such as a stent in order to remove, inhibit or prevent unwanted cellular proliferation or accumulation, in combination with an additional active agent.
  • Solid dosage forms for oral administration include but are not limited to, capsules, tablets, pills, powders, and granules.
  • the additional active agent, and/or the NTP peptide can be admixed with at least one of the following: (a) one or more inert excipients (or carrier), such as sodium citrate or dicalcium phosphate; (b) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (c) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (d) humectants, such as glycerol; (e) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (f) solution retarders, such as paraffin; (g) absorption accelerators, such as paraffin
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage forms may comprise inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers.
  • Exemplary emulsifiers are ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils, such as cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols, fatty acid esters of sorbitan, or mixtures of these substances, and the like.
  • oils such as cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, and sesame oil
  • glycerol tetrahydrofurfuryl alcohol
  • polyethyleneglycols fatty acid esters of sorbitan, or mixtures of these substances, and the like.
  • composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • compositions of the embodiments may be varied to obtain an amount of NTP peptide and additional active agent that is effective to obtain a desired therapeutic response for a particular composition and method of administration.
  • the selected dosage level therefore depends upon the desired therapeutic effect, the route of administration, the desired duration of treatment, and other factors.
  • body surface area may be approximately determined from the height and weight of an individual (see e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y. pp. 537-538 (1970)).
  • the total daily dose of the NTP peptide and additional active agent administered to a host may be in single or divided doses.
  • Dosage unit compositions may contain such amounts of such submultiples thereof as may be used to make up the daily dose. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of administration, potency of the administered drug, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated. It is preferred that the composition is administered only once as an injection or infusion, or in another preferred embodiment, the composition is administered twice. In this embodiment, the period of time between administration of the composition may vary anywhere from 2 months to 10 years, or from 8 months to 4 years, or more than about one year (e.g., between 1 and 2 years).
  • a method of administering an NTP peptide composition includes, but is not limited to, administering the compounds intramuscularly, orally, intravenously, intraperitoneally, intracerebrally (intraparenchymally), intracerebroventricularly, intratumorally, intralesionally, intradermally, intrathecally, intranasally, intraocularly, intraarterially, topically, transdermally, via an aerosol, infusion, bolus injection, implantation device, sustained release system etc.
  • Another method of administering an NTP peptide of the embodiments is by a transdermal or transcutaneous route.
  • the additional active agent may be employed together with the NTP peptide, or may be administered separately as discussed above, or may not be administered at all.
  • a patch can be prepared with a fine suspension of Peptide in, for example, dimethylsulfoxide (DMSO), or a mixture of DMSO with cottonseed oil and brought into contact with the skin of the tumor carrying mammals away from the tumor location site inside a skin pouch. Other mediums or mixtures thereof with other solvents and solid supports would work equally as well.
  • the patch can contain the Peptide compound in the form of a solution or a suspension.
  • the patch can then be applied to the skin of the patient, for example, by means of inserting it into a skin pouch of the patient formed by folding and holding the skin together by means of stitches, clips or other holding devices.
  • This pouch should be employed in such a manner so that continuous contact with the skin is assured without the interference of the mammal.
  • any device can be used which ensures the firm placement of the patch in contact with the skin.
  • an adhesive bandage could be used to hold the patch in place on the skin.
  • NTP peptides may be administered in a sustained release formulation or preparation.
  • sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules.
  • Sustained release matrices include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers, 22: 547-556), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed.
  • Sustained-release compositions also may include liposomes, which can be prepared by any of several methods known in the art (e.g., Eppstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692; EP 36,676; EP 88,046; and EP 143,949).
  • Another method of administering an NTP peptide of the embodiments is by direct or indirect infusion of the NTP peptide into the tumor or other tissue to be treated.
  • One example of such an embodiment is the direct injection of NTP peptide into the tumor or other tissue to be treated.
  • the treatment may consist of a single injection, multiple injections on one occasion or a series of injections over a period of hours, days or months with the regression or destruction of the tumor or other tissue to be treated being monitored by means of biopsy, imaging or other methods of monitoring tissue growth.
  • the injection into the tumor or other tissue to be treated may be by a device inserted into an orifice such as the nose, mouth, ear, vagina, rectum or urethra or through an incision in order to reach the tumor or tissue in vivo and may performed in conjunction with an imaging or optical system such as ultrasound or fibre optic scope in order to identify the appropriate site for the injection(s).
  • a device that can provide a constant infusion of NTP peptide to the tissue over time.
  • Another method of administering an NTP peptide is in conjunction with a surgical or similar procedure employed to physically excise, ablate or otherwise kill or destroy tumor or other tissue or cellular elements required or desired to be removed or destroyed wherein an NTP peptide of the embodiments is administered to the immediate area(s) surrounding the area(s) where the tumor or other tissue was removed in order to destroy or impede the growth of any tumor cells or other cellular elements not removed or destroyed by the procedure
  • Another method of administering an NTP peptide of the embodiments is by implantation of a device within the tumor or other tissue to be treated.
  • the additional active agent typically will be administered via a different route of administration than the NTP peptide.
  • One example of such an embodiment is the implantation of a wafer containing Peptide in the tumor or other tissue to be treated, and the administration of the additional active agent via oral administration. The wafer releases a therapeutic dose of NTP peptide into the tissue over time.
  • the composition may be administered locally via implantation into the affected area of a membrane, sponge, or other appropriate material on to which the NTP peptide has been absorbed.
  • the device may be implanted into any suitable tissue or organ, and delivery of the Peptide may be directly through the device via bolus, or via continuous administration, or via catheter using continuous infusion.
  • An alternative method of administration is to introduce one or more copies of an NTP peptide-encoding gene into the cell being targeted and, if necessary, inducing the copy(ies) of the gene to begin producing Peptide intracellularly.
  • the additional active agent typically will be administered via a different route of administration than the NTP peptide.
  • One manner in which gene therapy can be applied is to use the NTP peptide-encoding gene (either genomic DNA, cDNA, and/or synthetic DNA encoding the Peptide (or a fragment, variant, homologue or derivative thereof)) which may be operably linked to a constitutive or inducible promoter to form a gene therapy DNA construct.
  • the promoter may be homologous or heterologous to an endogenous Peptide-encoding gene, provided that it is active in the cell or tissue type into which the construct will be inserted.
  • Other components of the gene therapy DNA construct may optionally include, as required, DNA molecules designed for site-specific integration (e.g., endogenous flanking sequences useful for homologous recombination), tissue-specific promoter, enhancer(s) or silencer(s), DNA molecules capable of providing a selective advantage over the parent cell, DNA molecules useful as labels to identify transformed cells, negative selection systems, cell specific binding agents (as, for example, for cell targeting) cell-specific internalization factors, and transcription factors to enhance expression by a vector as well as factors to enable vector manufacture.
  • DNA molecules designed for site-specific integration e.g., endogenous flanking sequences useful for homologous recombination
  • tissue-specific promoter e.g., enhancer(s) or silencer(s)
  • DNA molecules capable of providing a selective advantage over the parent cell DNA
  • Means of gene delivery to a cell or tissue in vivo or ex vivo include (but are not limited to) direct injection of bare DNA, ballistic methods, liposome-mediated transfer, receptor-mediated transfer (ligand-DNA complex), electroporation, and calcium phosphate precipitation. See U.S. Pat. No. 4,970,154, WO 96/40958, U.S. Pat. No. 5,679,559, U.S. Pat. No. 5,676,954, and U.S. Pat. No. 5,593,875, the disclosures of each of which are incorporated by reference herein in their entirety.
  • a viral vector such as a retrovirus, adenovirus, adeno-associated virus, pox virus, lentivirus, papilloma virus or herpes simplex virus, use of a DNA-protein conjugate and use of a liposome.
  • a viral vector such as a retrovirus, adenovirus, adeno-associated virus, pox virus, lentivirus, papilloma virus or herpes simplex virus
  • gene therapy vectors is described, for example, in U.S. Pat. No. 5,672,344, U.S. Pat. No. 5,399,346, U.S. Pat. No. 5,631,236, and U.S. Pat. No. 5,635,399, the disclosures of each of which are incorporated by reference herein in their entirety.
  • the NTP peptide-encoding gene may be delivered through implanting into patients certain cells that have been genetically engineered ex vivo, using methods such as those described herein, to express and secrete the NTP peptide or fragments, variants, homologues, or derivatives thereof.
  • Such cells may be animal or human cells, and may be derived from the patient's own tissue or from another source, either human or non-human.
  • the cells may be immortalized or be stem cells. However, in order to decrease the chance of an immunological response, it is preferred that the cells be encapsulated to avoid infiltration of surrounding tissues.
  • the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow release of the protein product(s) but prevent destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues.
  • Methods used for membrane encapsulation of cells are familiar to the skilled artisan, and preparation of encapsulated cells and their implantation in patients may be accomplished without undue experimentation. See, e.g., U.S. Pat. Nos. 4,892,538; 5,011,472; and 5,106,627, the disclosures of each of which are incorporated by reference herein in their entirety.
  • a system for encapsulating living cells is described in PCT WO 91/10425.
  • An embodiment includes a method of reducing the incidence of acute urinary retention in mammals, including mammals suffering from BPH and/or any of the conditions listed in Tables 4-6, which comprises administering to the mammal at least once, a therapeutically effective amount of an NTP peptide, specifically an isolated peptide comprising the amino acid sequence in SEQ ID NO. 66 (Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu).
  • Another embodiment includes a method of reducing the incidence of acute urinary retention in mammals, including mammals suffering from BPH and/or any of the conditions listed in Tables 4-6, which comprises administering to the mammal a therapeutically effective amount of an NTP peptide, specifically an isolated peptide comprising the amino acid sequence in SEQ ID NO. 66 (Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu), followed by subsequent administration of one or more active ingredients known to be useful in treating acute urinary retention.
  • Another embodiment includes a method of reducing the incidence of acute urinary retention in a mammal that is not suffering from BPH, and in other embodiments, the mammal is suffering from one or more conditions selected from the group consisting of meatal stenosis, paraphimosis, penile constricting bands, phimosis, prostate cancer, organ prolapse selected from cystocele, rectocele, and uterine prolapse, pelvic mass selected from gynecologic malignancy, uterine fibroid, and ovarian cysts, retroverted impacted gravid uterus, aneurysmal dilation, bladder calculi, bladder neoplasm, fecal impaction, gastrointestinal or retroperitoneal malignancy mass, urethral strictures, stones, edema, balanitis, prostatic abscess, prostatitis, acute vulvovaginitis, vaginal lichen planus, vaginal lichen sclerosis, vaginal pemphigus, biharzi
  • the isolated peptide comprising the amino acid sequence in SEQ ID NO. 66 is administered in combination with at least one active agent selected from the group consisting of (1) of an inhibitor of 5 ⁇ -reductase and/or an antiestrogen, (2) an inhibitor of 5 ⁇ -reductase and/or an aromatase inhibitor, (3) a 5 ⁇ -reductase inhibitor and/or a 17 ⁇ -HSD inhibitor, (4) a 5 ⁇ -reductase inhibitor, an antiestrogen and an aromatase inhibitor, (5) a 5 ⁇ -reductase inhibitor, an antiestrogen and a 17 ⁇ -HSD inhibitor, (6) a 5 ⁇ -reductase inhibitor, an aromatase inhibitor, an antiestrogen and a 17 ⁇ -HSD inhibitor, (7) a 5 ⁇ -reductase inhibitor, an antiandrogen and an antiestrogen, (8), a 5 ⁇ -reductase inhibitor, an antiandrogen and an aromata
  • the isolated peptide comprising the amino acid sequence in SEQ ID NO. 66 is administered in combination with at least one active agent selected from the group consisting of alpha-adrenergic blockers such as alfuzosin or tamsulosin, dutasteride, tadalafil, terazosin, finasteride, doxazosin, combinations of finasteride and doxazosin, and mixtures thereof.
  • alpha-adrenergic blockers such as alfuzosin or tamsulosin, dutasteride, tadalafil, terazosin, finasteride, doxazosin, combinations of finasteride and doxazosin, and mixtures thereof.
  • compositions therefore are useful in treating a patient population different from only those suffering from BPH, and are useful in preventing or reducing the incidence of acute urinary retention for this patient population.
  • the compositions and methods are capable of reducing the incidence of acute urinary retention by an amount of more than about 60%.
  • compositions therefore are useful in treating a patient population different from only those suffering from BPH, and are useful in preventing or reducing the incidence of acute urinary retention for this patient population.
  • the compositions and methods are capable of reducing the incidence of acute urinary retention by an amount of more than about 50%.
  • patients with BPH were given an intraprostatic injection of PBS pH 7.2 vehicle alone or an injection of SEQ ID NO. 66 (Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu), 2.5 mg in PBS, under double-blind conditions by a urologist in an office setting under ultrasound guidance. Patients were followed for 1 to 6 years with regular physical examinations, laboratory tests, and evaluations of symptoms, and treatments.
  • NTP peptides of the present invention administered to patients requiring treatment for, or susceptible to developing, acute urinary retention, followed by subsequent administration of conventional oral medications used to treat BPH including alpha blockers such as tamsulosin, terazosin, doxazosin, or 5-alpha reductase inhibitors such as finasteride, dutasteride, or phosphodiesterase type 5 inhibitors (PDE5 inhibitors) such as tadalafil, would be expected to have an even more pronounced effect in preventing or reducing the incidence of acute urinary retention in patients susceptible to developing acute urinary retention.
  • the compositions therefore are useful in treating a patient population different from only those suffering from BPH, and are useful in preventing or reducing the incidence of acute urinary retention for this patient population.
  • NTP peptides of the present invention administered to patients requiring treatment for, or susceptible to developing, acute urinary retention, followed by subsequent administration of the NTP peptides or conventional oral medications used to treat BPH including alpha blockers such as tamsulosin, terazosin, doxazosin, or 5-alpha reductase inhibitors such as finasteride, dutasteride, or phosphodiesterase type 5 inhibitors (PDE5 inhibitors) such as tadalafil, would be expected to have an even more pronounced effect in preventing or reducing the incidence of acute urinary retention in patients susceptible to developing acute urinary retention, when compared to controls.
  • alpha blockers such as tamsulosin, terazosin, doxazosin, or 5-alpha reductase inhibitors
  • finasteride dutasteride
  • PDE5 inhibitors phosphodiesterase type 5 inhibitors

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US15/222,365 US10172910B2 (en) 2016-07-28 2016-07-28 Method of preventing or reducing the incidence of acute urinary retention
JP2019504756A JP6768140B2 (ja) 2016-07-28 2017-07-14 急性尿閉の発生を予防または低減させる方法
AU2017304100A AU2017304100B2 (en) 2016-07-28 2017-07-14 Method of preventing or reducing the incidence of acute urinary retention
ES17746197T ES2891544T3 (es) 2016-07-28 2017-07-14 Péptidos NTP para su uso en la prevención o reducción de la incidencia de la retención urinaria aguda
KR1020197006132A KR102489731B1 (ko) 2016-07-28 2017-07-14 급성 요정체의 발병을 예방하거나 감소시키는 방법
MX2019001209A MX2019001209A (es) 2016-07-28 2017-07-14 Metodo para evitar o reducir la incidencia de la retencion urinaria aguda.
EP17746197.7A EP3490580B1 (en) 2016-07-28 2017-07-14 Ntp peptides for use in preventing or reducing the incidence of acute urinary retention
RU2019105553A RU2721538C1 (ru) 2016-07-28 2017-07-14 Способ предупреждения возникновения или снижения частоты возникновения острой задержки мочи
PCT/IB2017/054277 WO2018020355A1 (en) 2016-07-28 2017-07-14 Method of preventing or reducing the incidence of acute urinary retention
CN201780046897.3A CN109562141A (zh) 2016-07-28 2017-07-14 预防或减少急性尿潴留发生的方法
CA3032254A CA3032254C (en) 2016-07-28 2017-07-14 Method of preventing or reducing the incidence of acute urinary retention
BR112019001750-0A BR112019001750A2 (pt) 2016-07-28 2017-07-14 método de prevenção ou redução da incidência da retenção urinária aguda
ZA201901176A ZA201901176B (en) 2016-07-28 2019-02-25 Method of preventing or reducing the incidence of acute urinary retention

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